Difference between revisions of "Team:Pittsburgh/Notebook"

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<h1 style="clear:both;">Week 2: May 31 - June 3</h1>
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<h1 >Week 2: May 31 - June 3</h1>
 
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<img src="https://static.igem.org/mediawiki/2016/b/b8/T--Pittsburgh--NotebookWetLab.jpg" alt="Wet Lab" style="padding:5px; width:100px;height:100px;float:left;">
 
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Revision as of 01:58, 2 August 2016

Our weekly progress. For a list of our protocols, visit the Protocols page

Week 1: May 23 - May 27

Wet Lab Dry Lab

Week 2: May 31 - June 3

Wet Lab

Cell-Free Extract

Dry Lab

Week 3: June 6 - June 12

Wet Lab

Reporter

  • Transform T7 promoter, amilCP, and terminator
  • Begin assembly by ligating linearized T7 promoter and amilCP
Dry Lab
  • Contact museums and summer programs for outreach opportunities

Week 4: June 13 - June 17

Wet Lab

Reporter

  • Transform T7-GFP plasmid, lacZ alpha fragment, and eGFP
  • Send promising T7 promoter -- amilCP ligations to be sequenced
  • Perform double digest of T7 promoter and terminator from last week
  • Ligate double-digested T7 promoter to new reporters (lacZ and eGFP)
Dry Lab
  • TECBio, DiSCoBio, and Camp BioE outreach opportunities set

Week 5: June 20 - June 26

Wet Lab

Cell-Free Extract

  • Reaction volume can be reduced to 5 μL with 5 ng/μL of DNA

Toehold Switch

  • Collins triggers activate the switches (both in plasmid form) to express LacZ

Reporter

  • Ligate T7 promoter -- amilCP construct to terminator
  • Extract successful ligations of T7 promoter to eGFP
  • Ligate T7 promoter -- eGFP construct to terminator
Dry Lab
  • Reach out to teams to collaborate based on last year's projects

Week 6: June 27 - July 3

Wet Lab

Cell-Free Extract

  • Reaction volume can be reduced to 1 μL with 5 ng/μL of DNA

Toehold Switch

  • Collins plasmids express LacZ with 25 ng of switch
  • DNA oligos trigger Collins switches

Reporter

  • Identify successful ligations to terminator for amilCP and eGFP constructs using a gel
  • Send correct plasmids for sequencing for confirmation
  • Test plasmids in cell-free extract
  • amilCP does not produce color in cell-free reaction
  • eGFP produces fluorescence comparable to that from the Collins T7-GFP plasmid in cell-free reaction
  • Linearized plasmids containing only the promoter and insert (no terminator) do not express protein
Dry Lab

Week 7: July 5 - July 8

Wet Lab

Cell-Free Extract

  • 384-well plate requires at least 10 μL of reaction

DNAzyme

  • Anneal PO strand with catalytic strand, both with and without erbium
  • Test success of annealing reaction in cell-free extract and with acrylamide gels

Reporter

  • Sequenced amilCP construct does not contain amilCP
  • Unsuccessfully linearize and amplify eGFP construct using PCR
Dry Lab
  • Practice outreach presentation for Camp BioE
  • Develop DNAzymes for other heavy metals

Week 8: July 11 - July 17

Wet Lab

Cell-Free Extract

  • Linear eGFP construct does not produce a stronger signal than its plasmid form

DNAzyme

  • DNAzyme duplex does not trigger toehold switch
  • Erbium cleaves the P substrate strand

Reporter

  • Restart amilCP cloning process
Dry Lab

Week 9: July 18 - July 22

Wet Lab

Cell-Free Extract

  • Reactions can be diluted by one-half and still produce visible results in two hours

DNAzyme

  • dPAGE assay of P substrate cleavage suggests that the DNAzyme works, but results are not definitive
  • Six-hour time course of cleavage does not yield much additional information
  • Reaction temperature (room temperature versus 37°C) does not produce observable effect on cleavage rates

Reporter

  • Continue amilCP cloning process

Amplifier

  • Clone RBS-T3 RNA polymerase to add into other constructs
Dry Lab

Week 10: July 25 - July 31

Wet Lab

Cell-Free Extract

  • Reactions diluted by one-half produce significantly less protein than undiluted reactions

DNAzyme

  • Annealing reactions produce hybrid complexes but leave unsequestered substrate strand
  • DNAzyme does not cleave P substrate strand

Reporter

  • Continue cloning amilCP construct
  • Determine sequence of possible lacZ plasmid
  • Unsuccessfully grow lacZ from iGEM bacterial stab

Amplifier

  • Continue cloning T3 constructs
Dry Lab
  • Presentation at Camp BioE
  • Start modeling toehold kinetics and the economical effects of lead

Week 11: August 1 - August 5

Wet Lab

DNAzyme

Reporter

Amplifier

Dry Lab
  • Presentation at Camp BioE
  • Meeting with Dr. Daniel Bain from the University of Pittsburgh Department of Geology and Environmental Science